Eustress and Malondialdehyde (MDA): Role of Panax Ginseng: Randomized Placebo Controlled Study.

Objective: The present study aimed at evaluating the effect of Panax Ginseng on malondialdehyde (MDA) serum levels during eustress on healthy volunteers. Method: In this study, 65 healthy volunteers were recruited from students of a medical school, with the mean age of 22.61±3.63 years. The volunteers were divided into 2 groups: Group A included 35 participants who were treated by Panax Ginseng 500 mg/day, which was regarded as the treated group; group B included 30 participants treated by placebo 500 mg/day, which was regarded as the control group. Baseline data were obtained and then one month after the study, the participants were followed with respect to induction of psychological stress through daily psychomotor performance task and visual working memory accuracy testing. Stress was assessed by malondialdehyde (MDA) serum levels. Results: The participants in the control group showed significant increases in MDA serum levels (p = 0.0004), which were related to significant increases in perceived stress scale from p<0.0001, while Panax Ginseng led to significant reduction in MDA serum levels (p<0.01), with significant increase in perceived stress scale (p = 0.02). Conclusion: Panax Ginseng produced significant reduction in oxidative stress and augmented eustress level in healthy volunteers 1 month after therapy.

Augustyniak et al. experimental study revealed that central nervous system is highly susceptible to the oxidative stress due to high brain metabolic and neuronal membrane that contains a large amount of fatty acids oxidized by free radicals. Cerebrospinal fluid contains a large amount of ascorbic acids and iron that are regarded as a source of toxic free radicals (8) . Psychological stress due to external psychomental stimuli is linked with stimulation of malondialdehyde (MDA) production due to triggering oxidative free radical formations. Mental stress in medical students during examination contributes to the induction of oxidative stress and elevation of MDA serum levels. Thus, overwhelming psychological stimuli activate neuronal oxidative phosphorylation at mitochondrial site, leading to imbalance between pro-oxidant and antioxidant levels, causing profound lipid peroxidation (9,10). Moreover, cognitive impairment is correlated with high free radical generations and low antioxidant capacity, indicating an association between the neuropathology and oxidative stress (11). Furthermore, oxidative stress markers are malondialdehyde (MDA), metalloenzymes, and selenium dependent glutathione peroxidase (12). However, MDA is regarded as a significant intermediate of hydroxyl radical, causing neuronal dysfunction and degeneration as MDA is a serious neuronal toxin (13). Brain oxidative stress is normally ameliorated and eliminated by free radical scavenger mechanisms including superoxide dismutase and glutathione. Thus, administration of Panax Ginseng or other antioxidants leads to significant activation of antioxidant activity and reduction of MDA serum levels (14). Therefore, the present study aimed at evaluating the effect of Panax Ginseng on malondialdehyde (MDA) serum levels during eustress on n healthy volunteers.

Materials and Methods
This study was conducted in Department of Clinical Pharmacology and Therapeutic, College of Medicine, Al-Mustansiriyia University from March to August 2016. The study was permitted and approved by Ethical Committee and Scientific Jury in the College of Medicine, Al-Mustansiriyia University.

Study Design
In this single blind study, 65 healthy volunteers were recruited from medical students at the School of Medicine with the mean age of 22.61±3.63 years (29 females and 36 males). The volunteers were divided into 2 groups. Group A: In this group, 35 participants (20 males and 15 females) were treated by Panax Ginseng 500 mg/day; this group was regarded as the treated group. Group B: In this group, 30 participants (16 males and 14 females) were treated by placebo (starch capsule) 500 mg/day; this group was regarded as the control group. Baseline data were obtained and 1 month after the study all volunteers in both groups were followed with respect to the induction of psychological stress by daily psychomotor performance task and visual working memory accuracy testing performed by the researchers. Psychomotor performance was estimated by Leeds Psychomotor Battery Tester, and visual working memory accuracy testing was estimated by Nback working memory test. These tests had 90% validity and reliability because of their intra-individual and inter-individual variations (15). Eustress was evaluated by Perceived Stress Scale (PSS) (16), while stress-induced oxidative stress was assessed by malondialdehyde (MDA) serum levels.

Assessment of Perceived Stress Scale (PSS)
PSS was done by a specific questioner that contained 14 items assessing the stress level of the enrolled healthy volunteers; the scales were 0 = never, 1 = almost never, 2 = sometimes, 3 = fairly often, and 4 = very often. Education level was assessed by a direct interview asking about the number of books read and general knowledge . Assessment of Malondialdehyde (MDA) Serum Levels Blood samples (5ml) were collected from each volunteer via vein puncture technique before and after the treatment to determine malondialdehyde (MDA) serum levels. Blood samples were located in plane tubes at room temperature to clot for 10 minutes, then, separated to get sera via centrifugation at 3000 / rpm for 20 minutes. The sera were stored at (-20•C), and MDA serum levels (nmol/ml) were estimated by ELISA kit method (Shanghai yehua biological technology Co., Ltd. China). The absorbance was read at 450 nm wavelength to calculate the concentration of standards and corresponding samples ( Figure 1).

Statistical Analysis
Data were presented as mean ± SD, numbers and percentages. Paired student t test was used to estimate the differences before and after treatment with either placebo or Panax Ginseng. Pearson Correlation coefficient was used to assess the correlation between perceived stress scale (PSS) and malondialdehyde (MDA) serum levels. P value was considered significant when it was less than 0.05 .

Results
In the present study, all volunteers continued the study without any withdrawal rate,; 55(84.61%) of the enrolled volunteers had a high education level, 6(9.23%) of them had a moderate education level, and 4 (6.15%) of them had low education level, . most Most of the enrolled volunteers were medical students 55(84.61%), while; 10(15.38%) of enrolled volunteers were medical students with extra work additionally; of the participants, 13(20%) of them were smokers, (table  Table 1 Table 3). Moreover, perceived stress scale was positively correlated with the oxidative stress as reflected by MDA serum levels (r = 0.693 significant, ly p<0.05 0.05) (figure Figure 2). Indeed, pPerceived stress scale was negatively correlated with augmented TRT. so;Thus as, as TRT values decreased the perceived stress scale will increased significantly (r = 0.691 p<0.05) (figure Figure 3).

Discussion
The present study demonstrated that frequent daily exposure to the positive and motivating psychological stressors leads to augmentation of perceived stress score and oxidative stress mediator (MDA) levels as oxidative stress like reactive oxygen species is not only linked with disease initiation and propagation, but it is also regarded as an important signaling messenger. Thus, removal of these oxidative mediators may be detrimental to the human health (17) . Oxidative stress naturally occurs during normal biological process that produces harmful reactive species, which attack the cellular membrane and constituents (18). Thus, mental and psychological stresses increase the risk of cognitive dysfunction due to reduction of natural antioxidant system. In addition, improvement in the psychomotor performance ability was related to decline in the oxidative stress and augmentation of endogenous antioxidant ability (19).
Padurariu et al. study demonstrated that MDA is a potential marker of lipid peroxidation during cognitive stress and that high MDA serum level is associated with oxidative damage that induced cognitive impairment (20). Panax Ginseng improves psychomotor performances and visual working memory with significant reduction in MDA serum levels and significant effect on perceived stress scale; these findings are supported by many studies that illustrated the potential role of Panax Ginseng in the treatment of cognitive and memory deficit (21), antioxidant effect (22), and anti-stress effect (23). Moreover,, the present study, showed that placebo produced insignificant effect on the psychomotor performances and visual working memory but with significant increments in MDA serum levels and perceived stress scale since placebo had insignificant antioxidant and anti-stress effects (24). This finding is supported by Asqaryet al. study that illustrated insignificant effect of placebo on oxidative stress and MDA serum levels in the evaluation of the effects of Hibiscus sabdariffa on oxidative stress in patients with metabolic syndrome (25). Additionally, this finding is sustained by Zolfaghari et al. research on unripe grape effect on the oxidative stress and blood pressure in rats that disclosed insignificant effect of placebo on the oxidative stress markers (26). In the present study, Panax Ginseng produced a significant effect on the reduction of MDA serum levels following oxidative stress induced by cognitive and psychological stresses by the ability of Panax Ginseng in the reduction of MDA serum levels. These findings are in agreement with various studies that revealed a potential effect of Panax Ginseng in reduction of MDA serum levels and stimulation of endogenous antioxidant enzyme given that Panax Ginseng improves cellular glutathione peroxidase and nitric oxide activity. Moreover, Panax Ginseng restrained ginsenosides and saponins that possess potent antioxidant activities via scavenging free radicals and inhibition of lipid peroxidation during acute psychological stress (27,28).
Moreover, Seo et al. study showed that compound K, isolated from Ginseng, possesses significant antioxidant effect, attenuate mitochondrial damage and cytotoxicity induced by glutamate during excitotoxicity induced by neuronal stimulation; it reverses scopolamine induced memory deficit through induction of Nrf2-mediated activity. Thus, Panax Ginseng therapy prevents neurological dysfunction caused by free radicals (29). Panaxynol derived from Ginseng is regarded as a potent activator of Nrf2 signaling, regulates cytokine expression, and has antioxidant activity (30). These findings may explain the effect of Panax Ginseng therapy in improving psychomotor performance, visual working memory accuracy, reduction of MDA serum levels, and augmentation of positive psychological stress (eustress), compared with placebo. They also signify the importance of eustress and rising in MDA serum levels without improvement in psychomotor performance and visual working memory accuracy; this indicates a positive correlation between MDA and stress scale as disclosed in the present study. Perceived stress scale was positively correlated with MDA serum levels as psychological eustress is associated with intensification of oxidative stress (31). However, total reaction time was negatively correlated with perceived stress scale, as augmented TRT was associated with high-perceived stress scale (32). Therefore, eustress or positive psychological stress was able to induce oxidative stress attenuated by Panax Ginseng therapy. Panax Ginseng in our study produced a mild significant effect on stress scale, which might have been due to the small sample size or significant antioxidant effect which decreased MDA induced eustress and adaptive response as MDA plays a role in autoregulation of cerebral blood flow during brain stimulation (33).

Limitations
First, this study had a single-blind technique, which might have biased the results. Second, gender differences were not estimated. Third, other stress scales were not estimated, so we could not make any comparisons among the scales. Fourth, only young participants were included in this study. All these factors might have limited our results. .

Conclusion
Panax Ginseng produced a significant reduction in oxidative stress and augmented eustress level in normal healthy volunteers one month after therapy